This invention relates to a cell potential measurement apparatus which is used in the field of electrical neurophysiology for measuring potential change associated with activities of nerve cells or nerve organs.
Recently, medical investigations into nerve cells and the possibility of using nerve cells as electric elements have been actively pursued. When nerve cells are active, action potential is generated. This action potential rises from a change in ion concentration inside and outside the cell membrane which is accompanied by a change in ion permeability in nerve cells and thus from the change in cell membrane potential accompanied thereby. Therefore, measuring this potential change accompanied by the ion concentration change (that is, the ion current) near the nerve cells with electrodes enables the detection of activities of nerve cells or nerve organs.
In order to measure the above-mentioned potential arising from cell activities, it is possible, for example, to insert an electrode comprising glass into an area of cells to measure extracellular potential. When evoked potential due to stimulation is measured, a metal electrode for stimulation is inserted together with a glass electrode for recording. However, measurement by the insertion of these electrodes has the possibility of damaging the cells, and measurement over a long period of time is difficult to carry out. In addition, due to restrictions of space and the need for operating accuracy, multipoint simultaneous measurements are also difficult to carry out.
Therefore, the present inventors developed a planar electrode comprising an insulation substrate and a multiplicity of microelectrodes and their drawer patterns formed thereon with the use of a conductive material, and cell culture could take place on that surface (disclosed in Laid-open Japanese patent application Nos. (Tokkai Hei) 6-78889 and 6-296595). With this planar electrode, multi-point simultaneous measurements of potential change can be carried out without being affected by restrictions of space at a plurality of points with a short electrode-to-electrode distance. Also, this electrode enabled long-term measurement.
However, a measurement apparatus which can efficiently use this kind of planar electrode, conduct measurements accurately and efficiently, and improve the arranging of measurement results has been strongly desired. Therefore, it is an object of this invention to provide a cell potential measurement apparatus which is capable of accomplishing these needs in the art.
In order to accomplish these and other objects and advantages, a cell potential measurement apparatus of this invention comprises (A) an integrated cell holding instrument provided with a plurality of microelectrodes on a substrate, a cell holding part for placing cells thereon, and an electric connection means for providing an electric signal to the microelectrodes and for leading out an electric signal from the microelectrodes; (B) a stimulation signal supply means to be connected to the electric connection means of the cell holding instrument for providing electric stimulation to the cells; and (C) a signal processing means to be connected to the electric connection means of the cell holding instrument for processing an output signal arising from electric physiological activities of the cells.
It is preferable that the cell potential measurement apparatus of this invention further comprises an optical observation means for observing the cells optically. It is also preferable that the cell potential measurement apparatus of this invention further comprises a cell culturing means for maintaining an environment for culture of cells which are placed on the integrated cell holding instrument. This configuration enables measurement over a long period of time.
Generally, the measurement conducted by means of the above-configured apparatus of this invention is carried out, for example, in the following steps. Sample cells are placed in a cell holding part of an integrated cell holding instrument, and a plurality of microelectrodes contact the cells.
An image of the cells is obtained by an optical observation means. A stimulation signal is applied between a pair of electrodes selected optionally from the plurality of microelectrodes by a stimulation signal supply means via an electric connection means. A change of evoked potential over time which is obtained in each of the other electrodes is provided to a signal processing means via the electric connection means, which is then output, for example, to a display device etc. after going through the necessary signal processing. The measurement of spontaneous potential which is not provided with a stimulation signal is carried out in a similar way.
The above-mentioned electric chemical measurement of cells must be conducted in a condition in which the cells are alive. Therefore, it is common to use cultured cells, and the cell holding part of the integrated cell holding instrument can be equipped with a culture medium. Since the integrated cell holding instrument is detachable from the measurement apparatus, each integrated cell holding instrument can be placed inside an ordinary incubator for cell culture and then taken out from the incubator and placed in the measurement apparatus. When a cell culturing means is further provided to maintain an environment for culture of the cells on the integrated cell holding instrument, long-term measurement is enabled. This cell culturing means comprises a temperature adjustment means for maintaining a constant temperature, a means for circulating a culture solution, and a means for supplying a mixed gas of air and carbon dioxide (e.g., CO2 5%).
It is preferable that the integrated cell holding instrument comprises a plurality of microelectrodes arranged in a matrix form (latticed) on the surface of a glass plate, conductive patterns for drawing these microelectrodes, electric contact points which are connected to edge parts of these conductive patterns, and a coating of insulation covering the surface of these conductive patterns, and the cell holding part is disposed in an area including the plurality of microelectrodes.
The use of a transparent glass plate as the substrate faciliates optical observations of the cells. Therefore, it is preferable that the conductive patterns or the insulation coating are also substantially transparent or translucent. Furthermore, when the plurality of microelectrodes is arranged in a matrix form, it is easier to specify positions of electrodes which are applied with stimulation signals or electrodes where voltage signals arising from cell activities are detected. For example, it is preferable to arrange 64 microelectrodes in 8 columns and 8 rows. In addition, the surface area of each electrode should be as broad as possible for reducing surface resistance and enhancing detection sensitivity. However, taking restrictions etc. arising from an electrode-to-electrode distance and space resolution of measurement into consideration, it is preferable that each electrode has a surface area of from 4xc3x97102 xcexcm2 to 4xc3x97104 xcexcm2.
Furthermore, it is preferable that the electric connection means includes a half-split holder which has a contact touching the electric contact point and fixes the glass plate by holding it from the top and bottom. According to this configuration, fixation of the glass plate and drawing of the microelectrodes to the outside can be performed easily and accurately. Furthermore, it is preferable that the electric connection means not only fixes the holder, but also comprises a printed circuit board having an outside connection pattern which is connected to the contact of the holder via a connector. As a result, connection with outside instruments, namely, with a stimulation signal supply means and a signal processing means is facilitated. For the transmission of stimulation signals or detection signals with as little attenuation and distortion as possible, contact resistance of the electric contact point with the contact as well as contact resistance of the contact with the connector are both preferably below 30 m ohm.
In addition, it is preferable that the optical observation means comprises an optical microscope, and an image pick-up device and an image display device connected to the optical microscope. In other words, the image of cells which is enlarged by a microscope is picked up by an image pick-up device (e.g., video camera) and then displayed in an image display device (e.g., a high-accuracy display), so that it is easier to conduct measurement while observing the cells and the electrode position. More preferably, when the optical observation means is further comprised of an image storage device, it is possible to record measurement results.
Also, when a pulse signal generator is used as the stimulation signal supply means, various kinds of signal waveforms can be applied as stimulation signals to the cells. It is preferable that the signal processing means comprises a multi-channel amplifier which amplifies a detection signal arising from cell activities and a multi-channel display device which displays an amplified signal waveform in real-time, and that signal waveforms (change of cell potential over time) obtained from a plurality of electrodes can be displayed simultaneously.
It is preferable that a computer is provided to output the stimulation signal via a D/A converter, and at the same time, to receive and process an output signal arising from electric physiological activities of the cells via an A/D converter. As a result, the stimulation signal can be determined as an optional waveform on the screen or a waveform of a detection signal can be displayed on the screen. In addition to these operations, it is easier to display these signals after being processed in various forms or to output them to a plotter or to store them. Furthermore, with the use of this computer, the optical observation means and the cell culturing means can be controlled.